The present invention relates to electrode membrane combinations for use in biosensors to detect analytes in a sample and to methods for the production of such electrode membrane combinations. The present invention also relates to methods for storing such electrode membrane combinations.
Biosensors based on ion channels or ionophores contained within lipid membranes that are deposited onto metal electrodes and where the ion channels are switched in the presence of analyte molecules have been described in International patent specification Nos WO 92/17788, WO 93/21528, WO 94/07593 and U.S. Pat. No. 5,204,239 (the disclosures of which are incorporated herein by reference). As is disclosed in these applications, ionophores such as gramicidin ion channels may be co-dispersed with amphiphilic molecules, thereby forming lipid membranes with altered properties in relation to the permeability of ions. There is also disclosure of various methods of gating these ion channels (for example, the lateral segregation mechanism disclosed in International Patent Application WO90/08783) such that in response to the binding of an analyte to a binding partner attached to the membrane, the conductivity of the membrane is altered. The applications also disclose methods of producing membranes with improved sensitivity using a surface amplifier effect, and improved stability and ion flux using chemisorbed arrays of amphiphilic molecules attached to an electrode surface. The applications further disclose means of producing lipid membranes incorporating ionophores on said chemisorbed amphiphilic molecules.
The present inventors have now determined improved means of producing electrode membrane combinations that result in sensor membranes with improved properties in terms of reproducibility, gating response towards an analyte, lateral segregation response, surface amplifier effect, stability in serum, plasma and blood, simplified production and the ability to store the membranes in a dry format (i.e. in the absence of any aqueous bath solution).
In the first aspect, the present invention consists in a method of producing a first layer electrode membrane comprising:
(1) Forming a solution containing Linker Lipid A (FIG. 1) the disulfide of mercaptoacetic acid (MAAD) or similar molecule, such as EDS linker Gramicidin B (FIG. 2), membrane spanning lipid C (MSL-C) (FIG. 3) and membrane spanning lipid D (MSL-D) (FIG. 3) or other linker molecules and ion channel or ionophore combinations as previously described:
(2) Contacting an electrode containing a clean gold surface with the solution, the disulfide containing components in the solution thus adsorbing onto the gold surface of the electrode;
(3) Rinsing the electrode with a suitable organic solvent; and
(4) Removing the excess organic solvent used for rinsing.
The nature of the membrane components are as follows:
Linker Lipid A comprising a benzyl disulfide attachment region, a hydrophilic region composed, in sequence, of tetraethylene glycol, succinic acid, tetraethylene glycol and succinic acid subgroups and an aliphatic chain;
The disulfide of mercaptoacetic acid (MAAD) or similar molecule, such as the disulfide of 2-mercaptoethanol (EDS).
Linker Gramicidin B is a linker molecule which comprises a benzyl disulfide attachment region, a hydrophilic region composed, in sequence, of tetraethylene glycol, succinic acid, tetraethylene glycol, succinic acid, and a hydrophobic region of gramicidin;
Membrane spanning lipid (MSL) D which comprises a benzyl disulfide attachment region, a hydrophilic region composed, in sequence, of tetraethylene glycol, succinic acid, tetraethylene glycol, succinic acid and a hydrophobic region of 1,1xe2x80x2dotriacontamethylenebis (2-3 RS,7R, 11-phytanyl) with an intermediate biphenyl region and a head group of phosphatidylcholine, hydroxyl, succinic acid, or PEG-400 COOH; and
Membrane spanning lipid C which comprises the same attachment and hydrophilic region as membrane spanning lipid D but differs in the head group which is a group consisting of (one to eight) 1,6-amino caproic acid and biotin.
In a preferred embodiment of the present invention the ratio of Linker Lipid A to the disulfide of mercaptoacetic acid (MAAD) or 2-mercaptoethoethanol (EDS) is 5:1 to 1:2, more preferably is 2:1.
It is further preferred that in order to improve the stability of the membrane, the amount of MSL-D in the first layer is as high as can be allowed and still maintain reasonable gramicidin conduction. The ratio of (Linker Lipid A+MAAD or EDS) to MSL-D is therefore preferably between 10:1 to 100:1.
In a further preferred embodiment, the amount of MSL-C is such that in the final sensor membrane an effective surface amplification on addition of analyte occurs, while still making it possible to suppress the lateral segregation induced gating on addition of the streptavidin, avidin or other similar biotin-binding protein. It should be noted that if the amount of MSL-C in the final sensor membrane is too large, then the excess protein that is bound to the MSL-C on addition of the streptavidin, avidin or similar biotin-binding protein will restrict the mobility of the gramicidin/receptor couple thereby reducing the gating response. In cases where the analyte molecule has multiple identical epitopes, MSL-C may capture the analyte molecules in preference to gramicidin/receptor couple, reducing the biosensor response.
It is therefore preferred that the ratio of (Linker Lipid A+MAAD or EDS) to membrane spanning lipid C is between 20,000:1 and 100:1.
It is further preferred that the ratio of (Linker Lipid A+MAAD or EDS) to MSL-C is 20,000:1.
As is known in the art, gramicidin exists in a monomer/dimer equilibrium in a bilayer membrane. In order for the gramicidin lateral segregation switch to function effectively, the ratio of monomer to dimer must be controlled. It is preferred that a proportion of the gramicidin ion channels exist as freely diffusing monomers in the outer membrane layer. The ratio of monomers to dimers can be controlled, amongst other methods, by changing the concentration of gramicidin in the first and second half of the membrane.
It is therefore preferred that the ratio of (Linker Lipid A+MAAD or EDS) to linker Gramicidin B is 10,000:1.
It is further preferred that the ratio of (Linker Lipid A+MAAD or EDS) to linker Gramicidin B is between 20,000:1 and 100,000:1 in those cases where it is necessary to minimise the amount of background leakage due to the adsorbed linker Gramicidin B.
It is preferred that the gold electrode consists of a freshly evaporated or sputtered gold electrode. It is further preferred that the gold electrode surface be freshly cleaned using a plasma etching process or an ion beam milling process.
It is preferred that the solvent for the adsorbing solution (step (1) and for the rinsing step (4) is ethanol.
In a second aspect, the present invention consists in a method of producing a monolayer electrode membrane comprising:
(1) Forming a solution containing the disulfide of mercaptoacetic acid (MAAD) or similar molecule (e.g. 2-mercaptoethanol (EDS)), membrane spanning lipid C(MSL-C) and/or membrane spanning lipid D (MSL-D) and, optionally, Linker Lipid A, linker Gramicidin B or other linker molecules or ion channel or ionophore combinations;
(2) Contacting an electrode containing a clean gold surface with the solution, the disulfide containing components in a solution thus adsorbing onto the gold surface of the electrode;
(3) Rinsing the electrode with a suitable organic solvent; and
(4) Removing the excess organic solvent used for rinsing, wherein the solution in step (1) contains more than a molar % of 50% of a membrane spanning lipid.
More preferably, the solution in step (1) contains more than a molar % or 70% of a membrane spanning lipid, 29% MAAD or EDS and 1% other membrane spanning lipids.
The preferred features and embodiments discussed above in regard to the method of the first aspect of the invention, may be equally applicable to the method of the second aspect of the invention.
The membranes produced by the method of the second aspect of the invention, do not form bilayers and have been found to be particularly resistant towards non-specific effects on addition of serum, plasma or whole blood to the sensor. Further advantages have been noted in that these membranes may be reused over a period of months in serum, plasma or whole blood without showing signs of degradation of performance. Monolayer lipid membranes are more practical for manufacturing purposes, have fewer manufacturing steps and greater stability, leading to a later expiry on the manufactured sensor containing such membranes. In this it is also preferable for the spacer molecule, MAAD or EDS, to be covalently linked to the membrane spanning lipids C or D, and covalently linked to PEPC, GDPE or triphytanyl PC, which increases stability of the final membrane.
A further preferred embodiment of the method according to the second aspect of the invention, consists in the use of valinomycin, covalently linked to the membrane spanning lipids C or D, via a linker of appropriate length such that the valinomycin is able to diffuse from one side of the membrane to another. This then results in a reusable biosensor, which does not need replenishment of the ionophore and could be used for an implantable device.
The present inventors have determined that the production of the biosensor is simplified and improved through the use of streptavidin, avidin or one of the related biotin bindingxe2x80x94proteins as a means of coupling a biotinylated receptor onto a biotinylated gramicidin ion channel or MSL.
In a third aspect, the present invention consists in a method of producing a second layer electrode membrane combination utilising biotinylated gramicidin E, in which the biotin is attached to the gramicidin via an amide to a lysine residue (preferred for chemical stability) or via an ester link to ethanolamine using a linker arm that is made up of between 1 to 8 aminocaproyl groups. The linker length, type, valency and number of linkers can affect the stability of the completed sensor and the optimum linker varies depending on the analyte being measured. The method comprises:
(1) Adding a solution of lipid and biotinylated gramicidin E (FIG. 4), dispersed in a suitable solvent onto the electrode surface containing a first layer produced as described in the first aspect of the present invention;
(2) Rinsing the electrode surface with an aqueous solution;
(3) Adding a solution of streptavidin, avidin, neutravidin, avidin or streptavidin derivative;
(4) Rinsing the electrode with an aqueous solution in order to remove excess streptavidin, avidin, neutravidin or other avidin or streptavidin derivative;
(5) Adding a solution of a biotinylated binding partner molecule; and
(6) Rinsing the coated electrode with an aqueous solution.
In a preferred embodiment of the present invention the lipid used in step (1) of the method of the third aspect is a mixture of diphytanyl phosphatidyl choline and glyceryl diphytanyl ether. The inventors have found that the combination of these lipids improves the stability of the bilayer membrane towards serum, plasma and whole blood, while still maintaining a good ionic seal, fluidity, reducing temperature effects on conduction and maintaining a true bilayer membrane structure.
It is further preferred that the diphytanyl phosphatidyl choline (DPEPC) and glyceryl diphytanyl ether (GDPE) is in a 7:3 ratio.
It is further preferred that the lipid is a triphytanyl phosphoryl choline as shown in FIG. (6).
It is also preferred that membranes contain 0 to 50%, more preferably 0 to 20% cholesterol in the second layer to enhance stability and analyte response in a serum, plasma or whole blood sample.
It is preferred that the ratio of lipid to biotinylated gramicidin E is between 10,000:1 and 1,000,000:1.
It is further preferred that the ratio of lipid to biotinylated gramicidin E is 100,000:1.
It is preferred that the biotin is attached to the gramicidin via the ethanolamine end using a linker arm that is between 10-80 angstroms long. It is preferred that the linker arm is hydrophilic.
It is preferred that the biotin is attached to the gramicidin via the ethanolamine end using a linker arm that is made up of between 1 to 8 aminocaproyl groups.
It is further preferred that two biotins are attached to the gramicidin via the ethanolamine end such that the biotins are able to bind simultaneously into the adjacent binding sites of one streptavidin, avidin or similar biotin-binding protein molecule, or into two separate streptavidin avidin or similar biotin-binding protein molecules. Alternatively, more than two biotin molecules can be attached to the gramicidin to produce multiple attachment sites for the binding partner molecules.
It is preferred that the two biotins are attached to the gramicidin via the ethanolamine end such that each biotin is attached to two to four linearly joined aminocaproyl groups that are attached to a lysine group as shown in FIG. (5). When more than two biotin molecules are attached to the gramicidin, a longer linker up to twenty aminocaproyl groups may be necessary these may be organised linearly or as a branched structure.
It is further preferred that in order to optimise the analyte response, it is necessary to minimise the signal caused by the presence of the linker. Thus, the amount of streptavidin, avidin or other similar biotin-binding protein that is added in step (3) is sufficient to cause a prozone effect, allowing most of the available biotinylated species in the membrane to have one streptavidin or related molecule bound to prevent crosslinking between gramicidin channels and MSL until a sample containing analyte is added to the sensor.
It is further preferred that prior to the addition of the streptavidin, avidin, or similar biotin-binding protein the lipid membrane electrode assembly is cooled. This reduces the fluidity of the membrane, decreasing the mobility of membrane components thus allowing the streptavidin, avidin or other similar biotin-binding protein to more readily bind to the biotinylated Gramicidin E and the membrane spanning lipid C without crosslinking between gramicidin channels and MSL until a sample containing analyte is added to the sensor.
It is preferred that the lipid membrane electrode is cooled to between 0xc2x0 and 50xc2x0 C., more preferably 0xc2x0 and 5xc2x0 C. It is further preferred that the subsequent rinsing and addition of the biotinylated binding partner molecule are also carried out at 0xc2x0 to 50xc2x0 C., more preferably 0xc2x0 to 5xc2x0 C.
It is preferred that the binding partner molecule is a biotinylated antibody or biotinylated antibody fragment.
It is further preferred that the binding partner molecule is a Fabxe2x80x2 fragment that is biotinylated via the free Fabxe2x80x2 thiol group.
Is further preferred that the linker between the Fabxe2x80x2 and biotins is between 10-80 angstroms in length. Is further preferred that the linker between the Fabxe2x80x2 and biotins consists of one to eight aminocaproyl groups.
It is further preferred that the group containing two biotins is attached to the antibody or antibody fragment such that the two biotins are able to complex simultaneously one streptavidin, avidin or other similar biotin-binding protein or two adjacent streptavidin, avidin or other similar biotin-binding protein molecules.
Alternatively, more than two biotins may be attached to the antibody or antibody fragment.
Furthermore, the present inventors have determined that by producing a covalently or passively coupled conjugate between the binding partner molecule and the streptavidin, avidin or other similar biotin binding protein the production of the biosensor membrane is further simplified.
Accordingly, steps 3 to 5 of the method of the third aspect can be substituted with:
(3) Adding a solution containing a conjugate between streptavidin, avidin, neutravidin or other avidin or streptavidin derivative and a molecule which is a member of a binding pair.
It is preferred that the binding partner molecule is an antibody or an antibody fragment such as an Fab or Fabxe2x80x2 or Fv fragment. Other binding pairs, which could be used in this invention would include: naturally occurring binding proteins and cellular receptors/analytes, enzymes or enzyme analogues/substrates, lectins/carbohydrates, complementary nucleic acid sequences and Anti-FC, Protein A or Protein G/antibody.
In order to manufacture sensor membranes efficiently and reproducibly, it is advantageous to incorporate the ionophore separate to the assembled membrane. It is also advantageous to bind one binding partner to the ionophore, before incorporation into the membrane. This both controls and enhances the reproducibility of membrane conduction and allows the reproducible attachment of the second binding partner needed in a two site immuno- or similar assay system, ensuring that only the first binding partner is attached to ionophore and only the second binding partner is attached to a second ionophore or MSL.
The present inventors have found that it is possible to co-disperse the hydrophobic ionophore in aqueous solution by several means, including:
1. The presence of a detergent, preferably at levels below the critical micelle concentration of the detergent, such that ionophore and the detergent form aggregates which allow the ionophore to remain in solution;
2. Conjugation of the gramicidin or other ionophore to a large molecular weight water soluble species; and
3. Attachment of the ionophore to a bead.
Furthermore it was found that it was possible to incorporate the functional ionophore into the biosensor lipid membranes by adding an aqueous solution of the ionophore/detergent aggregate to the solution bathing the preformed lipid biosensor membrane. This method of addition of the ionophore allows for a more controlled and reproducible method of incorporation of the ionophore into the lipid membrane.
Accordingly, in a fourth aspect, the present invention consists in a method of producing a second layer electrode membrane combination comprising:
(1) Adding a solution of lipid dispersed in a suitable solvent onto the electrode surface containing a first layer produced as described in the method of the first aspect of the present invention;
(2) Rinsing the electrode surface with an aqueous solution;
(3) Adding an aqueous solution containing ionophore co-dispersed with detergent or solubilised by coupling to a high molecular weight soluble species;
(4) Rinsing the electrode with an aqueous solution; and
(5) Adding the receptor using either streptavidin, avidin, or other similar biotin-binding protein followed by addition of a biotinylated antibody or antibody fragment or adding a streptavidin, avidin or similar biotin-binding protein conjugated to an antibody or antibody fragment as detailed in the third aspect of the present invention.
In a preferred embodiment of the present invention the lipid used in step (1) is a mixture of diphytanyl phosphatidyl choline and glyceryl diphytanyl ether. It has been found that the combination of these lipids improves the stability of the bilayer membrane towards serum, plasma and whole blood, while still maintaining a good ionic seal, fluidity, reducing temperature effects on conduction and maintaining a true bilayer membrane structure.
It is further preferred that the diphytanyl phosphatidyl choline and glyceryl diphytanyl ether is in a 7:3 ratio.
It is further preferred that the lipid is a triphytanyl phosphoryl choline as shown in FIG. (6).
It is also preferred that membranes contain 0 to 50%, more preferably up to 20% cholesterol in the second layer to enhance stability and analyte response in a serum, plasma or whole blood sample.
It is preferred that the aqueous solution used in step (3) of the fourth aspect of the present invention, contains gramicidin or a gramicidin derivative that is added to an aqueous solution of a detergent such that the detergent is present in excess relative to the gramicidin but that the total concentration of detergent is below the critical micelle concentration (CMC). The total detergent concentration is preferably kept below the CMC in order to minimise or negate any possible disruption of the membrane by the detergent.
The gramicidin/detergent solution is then preferably sonicated using an ultrasonic bath or horn for 5 to 20 minutes.
Preferred detergents are sodium dodecylsulfate, octylglucoside, tween, or other ionic or non-ionic detergents.
It is further preferred that the alkyl chain contains at least 7 or more methylene groups.
It is preferred that the detergent is sodium dodecylsulfate.
It is further preferred that the concentration of the sodium dodecylsulfate is less than 0.00001M and that the concentration of gramicidin is ten times less than the sodium dodecylsulfate concentration.
It has been found that, if it is necessary to store the electrodes which already have the first layer of the membrane adsorbed onto the electrode surface, then it is advantageous to store said electrodes covered in a solvent. This method of storing the electrode with the first layer membrane in a solution has been found to produce subsequent sensor membranes with improved homogeneity and ionophore gating ability, compared with storing the electrode in air.
Accordingly, in a fifth aspect the present invention consists in a first layer membrane electrode combination comprising an electrode and a first layer membrane comprising a closely packed array of amphiphilic molecules and a plurality of ionophores, the first layer membrane being connected to the electrode by means of a linker group as described previously, said first layer membrane being stored in the presence of a solvent.
Electrodes may be stored in a gaseous or liquid environment and, in a preferred embodiment, the solvent in which the electrodes are stored is an organic solvent or an aqueous solvent.
If the solvent is an organic solvent, it is further preferred that the solvent is an alcohol such as ethanol, glycerol, ethylene glycol, an alcohol or diol containing between 3 to 12 carbon atoms.
It is further preferred that the solvent is a hydrocarbon with between 8 to 20 carbon atoms. It is further preferred that the solvent is an aqueous solution containing a detergent.
It is further preferred that the solvent is a compound that is able to coat the electrodes such that oxidation of the electrode surface is minimised. It is preferred that such a solvent can be applied as a thin film.
Additionally it has been found that it is possible to store the complete sensor membrane electrode combination in a non-aqueous format. This is highly advantageous in terms of ease of manufacturing, shipping and storing of the biosensor product.
Accordingly, in a sixth aspect, the present invention consists in a lipid membrane based biosensor comprising a lipid membrane incorporating ionophores, the conductivity of the membrane being dependent on the presence or absence of an analyte, wherein the aqueous bathing solution in which the biosensor normally resides, is removed in a manner such that, on drying of said lipid membrane biosensor, the lipid membrane and the receptor molecules retain their function, structure and activity, when rehydrated.
It is preferred that in the drying process that the biosensor membrane does not have contact with the air-water interface, hence methods of drying such as lyophilisation, evaporation, or evaporation over controlled humidity, are recommended. It is also preferred that the concentration of the water-replacing agent is sufficient to protect all components within the membrane, i.e. lipid, ionophore and protein, during the drying process, during the storage time, and yet is easily removed upon the first addition of analyte or sample in the appropriate matrix, such that full activity of the biosensor membrane is restored immediately.
The water replacing substance may be either a protein, a low molecular weight diol or triol, a polyethylene glycol, a low molecular weight sugar, a polymeric peptide, polyelectrolyte or combinations of these substances, all of which are well known in the art. The main attributes of the water substitute are that it is highly polar, has a low vapour pressure, allows the membrane to retain its structure, is protein compatible and does not impede biosensor function when rehydrated. These substances may also be covalently bound to a specific membrane component, preferably a membrane spanning lipid.
It is preferred that the water replacing substance is bovine serum albumin, serum, fish gelatin, non-fat dry milk powder, casein, glycerol, ethylene glycol, diethylene glycol, polyethylene glycol, trehalose, xylose, glucose, sucrose, dextrose, ficoll and it is further preferred that the water replacing molecule is glycerol, sucrose, dextran or trehalose.
Such classes of molecules may also have the additional advantage in the biosensor to act as a spreading layer for serum/blood/analyte fluid addition; as a filter against specific cells, bacteria, virus particles, or classes of molecules; or as a reservoir containing specific displacement reagents required to compete off small analytes from proteins to which they are bound in serum or blood.
A further advantage of the water substituting agent is that it allows for the controlled rehydration of the lipid membrane without the lipid bilayer being in contact with the air/water interface as the analyte solution or sample is added.
An example of the latter is given in FIG. 13, where water-replacing molecules are either added or covalently bound to regions of the membrane and contain, for example, ANS (8-anilino-1-naphthalene-sulfonic acid) which competes with thyroxine for binding sites in albumin and thyroxine-binding globulin (TBG), releasing thyroxine for subsequent detection by the biosensor membrane.xe2x80x9d
The invention is hereinafter further described with reference to the following non-limiting examples and accompanying figures.